This proposal involves the analysis of chromosome structure and replication in the simple eucaryote yeast (Saccharomyces cerevisiae). Physical maps of two yeast chromosomes, chromosomes III and I, will be constructed. Various genetic and physical loci will be located on these maps and studied in detail. The replication and transcription of these chromosomes during the cell cycle will also be examined. The proposed research will be done in several steps: 1) Recombinant DNA molecules will be constructed by inserting randomly sheared yeast DNA into bacterial plasmids. These molecules will be used to transform E. coli, generating "clones" of bacteria. Each clone will contain a different segment of the yeast genome. 2) One or more clones containing DNA from a specific yeast chromosome (III or I) will be identified. 3) Once a single clone containing DNA from chromosome III or I is located, clones containing adjacent DNA sequences on the yeast chromosome can be identified by DNA-DNA hybridization experiments. By repeating this procedure several times with different probes, a physical map of a chromosome can be generated. All of the DNA from an average yeast chromosome could be contained in about 50 to 100 overlapping recombinant plasmids. 4) A number of loci (DNA initiation sites, genetically mapped loci, the centromere of the chromosome, etc.) will be located on the physical map and characterized. The recombinant plasmids will also be used to study DNA replication and transcription in yeast.